3rd Conference on Retroviruses and Opportunistic Infections


Washington, DC - January 28-February 1, 1996

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SINGLE TUBE ASSAY FOR THE QUANTITATIVE DETECTION OF HIV-1 RNA IN PLASMA SPECIMENS.

Conf Retroviruses Opportunistic Infect 1996 Jan 28-Feb 1; 3rd:60 (abstract no. 39)

Billyard B, Yang Y, Nunomura K, Garduno F, Clark T, Ueding K, Brentano S, Giachetti C, McDonough S, Mimms L
Gen-Probe Incorporated, San Diego, CA.

Quantification of plasma RNA levels in HIV infected patients has become an important tool in the study of HIV disease. Measurement of viral burden may be useful in monitoring a patient's disease progression or response to antiretroviral therapy. We have developed a novel quantitative detection methodology for the quantification of HIV-1 RNA in an assay in which specimen processing, amplification and detection are performed in a single tube.

In the single tube assay, genomic RNA is isolated from a plasma specimen in a simple procedure that does not involve the use of toxic chemicals and requires no centrifugation. Transcription Mediated Amplification (TMA) is used to amplify RNA target in a rapid, isothermal, transcription-based target amplification reaction which uses reverse transcriptase and RNA polymerase. Amplified product is detected using the Hybridization Protection Assay (HPA) a homogeneous hybridization and detection system in which unhybridized probe is selectively destroyed. No amplicon dilutions are needed. Specific primers targeted to sequences conserved among known HIV types (including type O) were utilized and sequences providing the preferred amplification range identified. Negative plasma was spiked with cultured HIV to obtain mock specimens of various titers. Using the single tube assay we demonstrated quantitation in the range of 20 to 200,000 copies of genomic RNA. The single tube assay can be completed in a few hours without expensive, specialized equipment.

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